This proposal involves a broad array of analytical studies designed to reveal new information about the virulence of newly emerged Vibrio cholerae variant strains of Vibrio cholerae (the causative agent of cholerae) and its Type VI secretion system (T6SS). The PI proposes to use state-of-the-art deep sequencing- based methods of transcript abundance (RNA-seq), mutant fitness (Tn-seq), and regulatory protein DNA binding site determination (ChIP-seq) to understand the hypervirulence and hyperinfectivity phenotypes of the H1 strain from the 2010 Haiti epidemic. He further proposes to study the biological activities of cyclic-AMP- GMP in promoting intestinal colonization of V. cholerae and hyperinfectivity by studying chemotaxis suppressor mutations and proteins that specifically bind or hydrolyze c-AMP-GMP. Because H1 is elevated in T6SS expression in vitro and other 7th pandemic strain express T6SS in vivo in infected rabbits, the PI also proposes to continue his highly productive investigations on the function of the dynamic T6SS organelle. His lab will test the hypothesis that T6SS spike tip PAAR proteins are adapters for transport of multiple T6SS effector domains into target bacterial and eukaryotic cells by V. cholerae by driving their association with the VgrG trimer of the T6SS spike. A high resolution mutation analysis technology developed in the PI's laboratory (Mut-Seq), will be used to obtain a deeper understanding of the dynamic V. cholerae T6SS apparatus by defining mutations that are compatible or incompatible with various models for how the organelle functions. Moreover, he will apply Mut-seq to understand the functionality of V. cholerae T6SS proteins by defining dominant negative mutations that block T6SS function. These mutations may provide the tools to capture and stabilize intermediates in the T6SS assembly/firing cycle that will aid in fine structure analysis as well as inform efforts to develop drugs that block the function of the T6SS organelle. Because this grant has previously funded cholera vaccine development efforts in the PI's lab, he also proposes to continue research on new live attenuated and nanoparticle-based vaccines derived from V. cholerae and its T6SS, respectively. These include a promising nonreactogenic, nonflagellated derivative, Peru-NT FlaABCDE as well as multivalent nanoparticle derived from the T6SS sheath. He proposes to test whether Hcp and VgrG proteins are recognized by the innate immune system and thus potentially act as adjuvants in vaccine formulations, as well as test the possibility that the V. cholerae T6SS can delivery CTL epitopes to the host cell cytosol and thus stimulate CD8 T cell responses against heterologous antigens. Finally, the PI proposes to develop new technologies that will allow facile phenotypic analysis in V. cholerae and other organisms. These include 'Sortase conjugation technology' (to study protein localization and trafficking in living cells) as well as combinatorial J25 microcin 'lasso peptides' (to develop drug-like small molecule inhibitors of T6SS function). These technologies represent promising and powerful tools to understand more about the T6SS organelle of V. cholerae and its anti-cellular effectors.